In order to unravel gene regulatory pathways in eukaryotic cells, it is necessary to achieve a much greater understanding of the basic RNA synthesis machinery. In particular, it is now appreciated that control of transcript elongation is an important aspect of the regulation of gene expression. The DNA template for transcription in the cell nucleus is packaged into nucleosomes. However, during in vitro transcription experiments nucleosomes are usually very strong barriers to transcript elongation. Our long-term goal is to obtain an in vitro transcription system that more accurately reflects in vivo events. It is therefore necessary to much more fully characterize the molecular mechanisms through which RNA polymerase II transcribes nucleosomal templates. We will be guided by the limited number of examples in which some transcription of nucleosomal DNA has been achieved in vitro. For example, results with mononucleosomal templates have emphasized the potential importance of DNA adjacent to the nucleosome as an acceptor for nucleosomal DNA binding surfaces left unoccupied as RNA polymerase acquires the template DNA. We will therefore prepare polynucleosomal templates consisting of arrays of nucleosomes in defined locations. This will allow us to systematically explore the importance of polymerase-nucleosome and nucleosome-nucleosome spacers in the efficiency of transcript elongation. There is considerable evidence that modification of the N-terminal domains of histones is important in the control of gene expression. We will build on this finding, and on our own preliminary results, to explore the effect of modifications in the histone N-terminal domains on the ability of RNA polymerase to transcribe nucleosomal templates. Finally, recent work has identified a novel transcription factor, FACT, which specifically facilitates transcript elongation through nucleosomes. We will investigate the mechanism by which FACT functions, and we will also search for effects of other elongation factors on the transcription of nucleosomal templates.